Project Summary Hybrid vigor, also known as heterosis, refers to the increase in stature, biomass, and fertility of a hybrid that is superior to one or both parents. Hybrid vigor is a fundamental biological phenomenon that has translational impact on agriculture, food security, and human health. Heterosis is commonly observed in flowering plants and other sexually reproducing organisms including humans. In spite of its scientific significance and economic importance, molecular bases for heterosis are poorly understood. A recent breakthrough finding has linked altered circadian rhythms to growth vigor in Arabidopsis hybrids and allopolyploids. The altered circadian regulation in the hybrids increases expression of circadian-mediated genes in energy and metabolism, promoting growth vigor. In humans, disrupting circadian rhythms causes jet leg, diseases and physiological disorders. A key question remains: how and when does the genomic mixture in the hybrids alter gene expression and growth vigor? Several findings have led us to address this question. First, there is a parent-of- origin effect on altered circadian gene expression and hybrid vigor in embryos, which resembles a classical epigenetic phenomenon of imprinting. Second, consistent with the notion, chromatin modifications and RNA- directed or de novo DNA methylation mediate the parent-of-origin effect. Third, a circadian clock regulator and a histone deacetylase are found in the same complex, supporting an interactive role between chromatin and circadian regulation. A long-term goal of the project is to define mechanisms and processes for improving circadian rhythms and heterosis. Towards this end, experiments are designed to determine if de novo and/or maintenance DNA methylation is necessary and sufficient to regulate allelic gene expression and hybrid vigor and if maternally inherited small interfering RNAs move to regulate spatial gene expression in the embryo. Notably, the parent-of-origin effect is confounded by imprinting in nuclei and by maternal effect in the cytoplasm. These confounding effects will be discriminated using newly developed isogenic cytoplasmic- nuclear substitution lines (CNS); each pair contains the same cytoplasm and different nuclei or vice versa. The CNS lines will be used to make different hybrid combinations to identify the genes that are subject to imprinting or maternal effect. At the mechanistic level, there is evidence that clock regulators interact with chromatin factors. Genetic and biochemical approaches will be employed to determine how DNA methylation and chromatin factors are integrated into the circadian pathway that regulates hybrid vigor. Proteomic approaches will be used to identify new chromatin factors in hybrid embryos, and their functions will be elucidated. Results from these integrated approaches will provide new and mechanistic insights into the establishment of hybrid vigor in embryos. The principles for developmental and epigenetic regulation of circadian rhythms, imprinting, and maternal effect will be directly or indirectly relevant to animal development and human health.